International Research Journal of Biological Sciences ______ISSN 2278-3202 Vol. 1(4), 33-38, August (2012) I. Res. J. Biological Sci.

In-vitro Antimicrobial and Cytotoxic ativity of Methanolic extract of Osbeckia wynaadensis

Illath Sujina and Subban Ravi * Department of Chemistry, Karpagam University, Coimbatore-641 021,

Available online at: www.isca.in Received 6th July 2012, revised 14 th July 2012, accepted 16 th July 2012

Abstract In- vitro antibacterial and antifungal activities of methanolic extract of osbeckia wynaadensis was evaluated in the present study for the first time by disc diffusion method using five bacterial strains (S. pneumonia, B.cereus, A. hydrophila , V. cholera and MRSA) and five fungal strains (Candida albicans, Aspergillus niger, Streptomysis Greusis, M. purpureus and Aspergillus fumigate) respectively. The zone of inhibition and minimum inhibitory concentration were measured. Ampicillin (30 µg/disc) and clotrimazole (20 µg/disc) were used as standard for antibacterial and antifungal activity respectively. In- vitro cytotoxic activity of Osbeckia wynaadensis was evaluated against the human cervical adenocarcinoma cell line (HeLa) and murine embryonic fibroblasts cell line (NIH 3T3) by MTT assay and the IC 50 value found to be 220.3 µg/mL and 93. 25 µg/mL. It is concluded that Osbeckia wynaadensis exhibited significant antibacterial, antifungal & in-vitro cytotoxic activity.

Key words: Osbeckia wynaadensis , in-virto cytotoxicity, MTT assay, antibacterial activity, antifungal activity.

Introduction medicinal relevance with the recent years, infections have increased to a great extent and resistant against antibiotics, All over the world, people depended on the herbs for the becomes an ever increasing therapeutic problem 9. Natural treatment of various ailments before the advent of modern products of higher may give a new source of medicine. Medicinal plants constituents an arsenal of chemicals antimicrobial agents. There are many research groups that are that could be exploited by human to prevent microbial invasion. now engaged in medicinal plants research 10-13 . They have been a major source for drug development. extracts and products are used in the treatment of bacterial, Osbeckia wynaadensis (Melastomatacea), a herb, is distributed 1 fungal and viral infection . Plants play a vital role in our lives wild in Western Ghats, along river banks. The genus Osbeckia more than animals mainly due to their extraordinary array of contains about 12 species. The whole plants of this genus are diverse class of biochemicals with a variety of biological used as traditional medicine with the function of heat-clearing 2 activities . Plants have been used for the treatment of disease all and detoxicating, hematischesis and astrigence. And those roots over the world before the advent of modern clinical drugs. are used to treat dysentery and gonorrhea 14 . Chemical Natural phytochemicals are known to contain substance that can constituents were reported from O.crinita, O.aspera, be used for therapeutic purposes or as precursor for the O.chinensis showed the presence of flavanoids 15-17 , organic synthesis of novel useful drugs. The natural products play an acids 18 and steroids19 . The biological activities of the Osbeckia important role in drug development in pharmaceutical industry. genus are given in the table-1. Antioxidant and Use of plant as a source of medicine has been inherited and is an immunomodulatory effects were investigated with O.aspera and 3 important component of the health care system . O.octandra was studied for its hepatoprotective activity. Further O.octandra , O.chinensis and O.nepalensis were showed to Plants have a long history of use in the treatment of cancer. exhibit antidiabetic activity. Altogether so far six species has Drug discovery from plants is a multi ‐disciplinary approach been investigated and all other species was yet to explore for which combines various botanical, ethno ‐botanicals, their phytoconstituents and biological activities. This promoted photochemicals and biological and chemical separation us to investigate the remaining osbeckia species for its activities 4 techniques . However, despite these observations, it is and phytoconstituents. To start with O.wynaadensis was significant that over 60% of currently used anti ‐cancer agents selected for our present work. It is a slender erect under shrub are derived from natural sources, including plants, marine with purple flowers in sub- terminal corymbs, large long- 5,6 organisms and micro ‐organisms . petioled leaves and very characteristic comb-like scales on the calyx-tube. Plant pacifies vitiated pitta, inflammation, urinary Aromatic and medicinal plants are known to produce certain tract infection, hemorrhage, menorrhagia, hemorrhoids and bioactive molecules which react with other organisms in the leucorrhea. Presence of this plant is an indication of pure environment, inhibiting bacterial or fungal growth 7,8 . The underground water. This plant is on the verge of extinction due antimicrobial activity have been screened because of their great to water pollution and manmade destructive-activities.

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As far as our literature survey no work is reported on this plant. Antifungal screening: The inoculums for the experiment were The present work is to evaluate the antibacterial, antifungal and prepared in fresh sabouraud’s broth from preserved slant cytotoxic activity of the methanolic extract of O.wynaadensis. culture. The inoculum was standardized by adjusting the turbidity of the culture to that of McFarland standards. The Material and Methods turbidity of the culture may be adjusted by the addition of sterile saline or broth (if excessive or by further incubationg to get Collection and identification of plants: The whole plant of required turbidity (Leonard Jarrett et.al.). Cotton wool swab on O.wynaadensis is collected during March-April 2009, from wooden applicator or plastics were prepared and sterilized by Wayanad, Kerala, India. The plant was authetificated from BSI, autoclaving or dry heat (only for wooden swabs) by packing the Coimbatore and the specimen no is BSI/SRC/5/23/2010-11/Tec- swabs in culture tubes, papers or tins etc. The standardized 2098. inoculums is inoculated in the plates prepared earlier (aseptically) by dipping a sterile in the inoculums removing the Preparation of extract: The collected plants were washed with excess of inoculums by passing by pressing and rotating the water and dried in shade. The air dried plant is powdered and swab firmly against the side of the culture tube above the level 1.5 kg of this powdered material was soaked in 70% methanol of the liquid and finally streaking the swab all over the surface for 72 hours and the extract was collected and concentrated to of the medium 3 times rotating the plate through an angle of 60º yield a residue (50 mg). after each application. Finally pass the swab round the edge of the agar surface. Leave the inoculums to dry at room Microorganisms: The following bacterial strains were temperature with the lid closed. employed in the screening: Gram positive Streptococcus pneumonia and Bacillus cerus and the Gram negative Each Petri dish is divided into 2 parts, in 2 parts extract discs Aeromonas hydrophila and Vibrio cholera and Methicilin- such as osw (250mcg) discs, (discs are soaked overnight in resistant staphylococcus aureus (MRSA). In the antifungal extract solution) and one quadrant for Std clotrimazole 10mcg, screening the following fungi were tested: Candida albicans, are placed in each quadrant with the help of sterile forceps. Aspergillus niger, Streptomysis Greusis (mm), M.purpureus, Then Petri dishes are placed in the refrigerator at 4ºC or at room Aspergillus fumigate . temperature for 1 hour for diffusion. Incubate at room temperature for 24 - 48 hours. Observe the zone of inhibition Antibacterial screening: Disc diffusion method: The bacterial produced by different Antibiotics. Measure it using a scale or strains ( Streptococcus sp. , B.cereus, A. hydrophila V. cholerae divider or venire calipers and record the average of two were inoculated in the nutrient broth under aspectic condition diameters of each zone of inhibition. 0 and incubated at 37 C for 18 hours. After the incubation period, the test bacterial was swabbed on the nutrient agar plate using Cytotoxic Activity: The human cervical adenocarcinoma cell sterile cotton swab. In each of these plates, wells (10 mm) were line (HeLa) and murine embryonic fibroblasts cell line (NIH cutout using sterile cork borer. The methanol extract was 3T3) were obtained from National Centre for Cell Science dissolved in the solvent. Controls were maintained by loading (NCCS), Pune. HeLa was grown in Eagles Minimum Essential same quantity of Ampicillin into the wells. Then the petri dishes Medium (EMEM) and NIH 3T3 was grown in Dulbeccos 0 were incubated at 37 C for 14 hours. The anti microbial activity Modified Eagles Medium (DMEM) supplemented with 10% was evaluated by measuring the zone of inhibition in diameter. fetal bovine serum (FBS). All cells were maintained at 37 0 C, The zone of inhibition in diameter was observed and recorded in 5% CO2, 95% air and 100% relative humidity. Maintenance millimeter. The same method was carried out for MRSA using cultures were passaged weekly, and the culture medium was Ampicillin as the positive control. changed twice a week.

Minimum Inhibitory concentration (MIC): The Minimum The monolayer cells were detached with trypsin- inhibitory concentration (MIC) was determined through the ethylenediaminetetraacetic acid (EDTA) to make single cell 20 dilution method . Bacteria were grown in nutrient broth (NA) suspensions and viable cells were counted using a for 6 hrs. After this, 20 µL of 106 cells/mL were inoculated in hemocytometer and diluted with medium containing 5% FBS to tubes with nutrient broth supplemented with 5 different give final density of 1x10 5 cells/ml. one hundred microlitres per concentrations (25 µL, 50 µL, 100 µL, 150 µL, 200 µL) of the well of cell suspension were seeded into 96-well plates at oils. After 24 hrs at 37°C, the MIC of each sample was plating density of 10,000 cells/well and incubated to allow for 0 measured through optical density in the spectrophotometer cell attachment at 37 C, 5% CO 2, 95% air and 100% relative (620nm) through the comparison of the sample readout with the humidity. After 24 h the cells were treated with serial 21 known inoculated nutrient broth and the results are enlisted in concentrations of the test samples. They were initially dissolved tables, Ampicillin was used as a standard substance, DMSO as in neat dimethylsulfoxide (DMSO) and diluted to twice the the negative control. The same method was carried out for desired final maximum test concentration with serum free MRSA using Ampicillin as the positive control, DMSO as the medium. Additional four, 2 fold serial dilutions were made to negative control. provide a total of five sample concentrations. Aliquots of 100 µl

International Science Congress Association 34 International Research Journal of Biological Sciences ______ISSN 2278-3202 Vol. 1(4), 33-38, August (2012) I. Res. J. Biological Sci. of these different sample dilutions were added to the appropriate Anti MRSA activity: Methicillin-resistant Staphylococcus wells already containing 100 µl of medium, resulted the aureus (MRSA) has become endemic in most hospitals and required final sample concentrations. Following drug addiction health care facilities. The MRSA strains are broadly resistant to the plates were incubated for an additional 48 h at 37 0 C, 5% β-lactam and macrolide/azalide antimicrobials but responsive to 24,25 CO 2, 95% air and 100% relative humidity. The medium certain non-β-lactam antibiotics . However, resistance rates containing without samples were served as control and triplicate are increasing and there are other limitations in the use of those was maintained for all concentrations 22, 23 . drugs. Thus given the widespread dissemination and morality caused by MRSA, the synthesis and development of new drug is MTT assay: MTT is a yellow water soluble tetrazolium salt. A imperative. mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble In the present study methanolic extract of O.wynaadensis is too purple formazan. Therefore, the amount of formazan produced led for its MRSA activity. Our drug showed moderate activity is directly proportional to the number of viable cells. against the Methicillin-resistant Staphylococcus aureus. It showed zone of inhibition 14 mm, MIC at 2.5 mg. The standard After 48h of incubation, 15µl of MTT (5mg/ml) in phosphate compound Ampicillin showed 17 mm but the MIC at 8.0 mg. buffered saline (PBS) was added to each well and incubated at This is the first report on the antimicrobial activity of the 37 0C for 4h. The medium with MTT was then flicked off and O.wynaadensis and the Osbeckia genus. The results are given in the formed formazan crystals were solubilized in 100µl of table 4. DMSO and then measured the absorbance at 570 nm using micro plate reader. The % cell inhibition was determined using Overall the results suggest that the methanolic extract of the following formula. O.wynaadensis may be potential use in the treatment of MRSA % cell Inhibition = 100- Abs (sample)/Abs (control) x100. and other bacterial infections.

Nonlinear regression graph was plotted between % Cell Cytotoxic activity (MTT assay): O.wynaadensis showed inhibition and Log 10 concentration and IC50 was determined cytotoxic activity against HeLa and NH3T3 and the IC50 value using GraphPad Prism software. found 220.3 µg/mL and 92.35 µg/mL respectively. The results are given in the table 5. Results and Discussion In the present study, In-vitro cytotoxic effect of methanolic Antibacterial and antifungal Screening: Antimicrobial extract of O.wynaadensis against human cervical cancer cell activity was conducted against a food borne pathogenic line ( HeLa ), mouse embryonic fibroblasts cell line ( NIH 3T3 ) microorganisms including Gram positive and Gram negative cell lines were determined by MTT assay. The results indicate bacteria and fungi. that O.wynaadensis showed good activity against murine embryonic fibroblasts cell line ( NIH 3T3 ), IC 50 93.2 The antibacterial activity and antifungal activity of the extracts µg/mLcompared to human cervical cancer line IC 50 220 µg/mL. of O.wynaadensis at different concentrations was screened by This is the first report of its kind on the in-vitro cytotoxic disc diffusion technique and the zone of inhibition was activity against HeLa and NIH3T3 cancer cell lines. measured in mm diameter. The results are given in the table 2 and 3 respectively. Conclusion

The antimicrobial activity of the O.wynaadensis against gram In the present study we confirm the in-vitro cytotoxicity activity (+ve) and gram (-ve) bacteria shown in table 2. O.wynaadensis and antimicrobial activity of methanolic extract of exhibited inhibitory activity against S. pneumonia , B.cereus, A. O.wynaadensis was carried out for the first time. hydrophila, V. cholera with narrow inhibition zones of 16.0, 12.0, 12.0 and 14.0 mm and MIC value of 2.5, 2.0, 2.5, 2.0 Reference mg/ml and for MRSA an inhibition zone of 14 mm and MIC value of 2.5 mg/ml. 1. Bruneton J., Pharmacognosie: Phytochime, Plants medicinal, Tec and Doc, Paris, 309-354 (1999) The methanol extract exhibited significant antifungal activity 2. Cotton C.M., Ethanaobotony: Principals and Applications, against most of the tested fungi species with zones of inhibition John Wiley and Sons Ltd. Chichister, England (1996) between 11-25 mm at the tested concentration. The antifungal activity of O.wynaadensis against Candida albicans, Aspergillus 3. Ahmedulla M. and Nayar M.P., Red Data Book of Indian niger, Streptomysis Greusis, M.purpureus and Aspergillus Plants, Calcutta; Botanical Survey of India, 4, (1999) fumigate with narrow inhibition zone 11.0, 12.0, 16.0, 11.0 and 4. Jachak S.M. and Saklani A., Challenges and opportunities 25.0 mm and MIC value of 2.5, 2.5, 1.25, 2.5, 1.25 mg/ml and in drug discovery from plants, Current science, 92(9 ), the results are comparable with the standard substance. 1251 ‐57 (2007)

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5. Cragg G.M., Kingston D.G.I. and Newman D.J. (Eds.), Stockholm University, SE-106 91 Stockholm, Sweden Anticancer Agents from Natural Products Boca Raton, Limnol. Oceanogr ., 53(6), 2636–2643 (2008) Florida: Taylor and Francis (2005) 20. Chlebowski R.T., Grosvenor M., Lillington L., Sayre J. 6. Singh Shachi, Antimiotic activity of a New Compound and Beall G., Dietary intake and counseling, weight Isolated from the Flower of Prosopis juliflora, Research maintenance, and the course of HIV infection, J Am Diet Journal of recent Science 1(6), 22-26 (2012) Assoc, 95(4) , 428-435 (1995) 7. Chopra R.N., Nayer S.L. and Chopra I.C., Glossary of 21. Mossman T., Rapid colorimetric assay for cellular growth Indian medicinal plants, 3 rd Edn. New Delhi: Council of and survival: application to proliferation and cytotoxicity Scientific and Industrial Research, 7(1), 239-46 (1992) assays, J of Immunological Methods , 65 , 55-63 (1983) 8. Bruneton J., Pharmacognosy, phytochemistry, medicinal 22. Monks A., et al, Feasibility of high flux anticancer drug plants. France: Lavoisiler Publishing Co., 265-380 (1995) screen using a diverse panel of cultured human tumour cell J. of the National Cancer Institute 83 9. Austin D.J., Kristinsson K.G. and Anderson R.M., The lines, , , 757-766 relationship between the volume of antimicrobial (1991) consumption in human communities and the frequency of 23. Patel M., Community-associated methicillin-resistant resistance, Proc Natl Acad Sci USA, 96, 1152-6 (1999) staphylococcus aureus infection: epideminology, 69, (2009) 10. Samy R.P., Ignacimuthu S. and Sen A., Screening of 34 recognition and management, Drug 693-716 Indian medicinal plants for antibacterial properties, 24. Powell J.P. and Wenzel R.P., Antibiotic option for treating Journal of Ethanopharmaco logy, 62(4), 173-81 (1998) community-acquired MRSA, Expert Rev. Anti Infect. Ther . 6 (2008) 11. Mangale Sapna M., Chonde Sonal G. and Raut P.D., Use , 299-307 of Morigna Oleifera (Drumstick) seed as natural 25. Jayatilaka K.A. and Thabrew M.I., Absorbent and an Antimicrobial agenr for Ground water against carbon tetrachloride-mediated alterations in treatment, Research Journal of recent Science, 1(3), 31-40 microsomal drug metabolizing, J Pharm Pharmacol , (2012) 52(4) , 461-5 (2000) 12. Hedge Chatra R., Madhuri M., Swaroop T., Nishitha Das 26. Ira Thabrew M., Robin D., Hughes and Ian G. McFarlane, Arijit, Bhattacharya Sourav and Rohit, Evaluation of Antioxidant activity of Osbeckia aspera, Phytotherapy Antimicrobial Properties, Phytochemical Content and research, 12(4), 288-290 (1998) Punica Antioxident Capacities of Leaf Extracts of 27. Renee J. Gayer M., Ira Thabrew, Robin D., Hughes Sam granatus L., ISCA Journal of Biological Science, 1(2) , 32- Bretherton, Andrew Lever, Nigel C. Veitch, Geoffrey C. 37 (2012) Kite, Roberto Lelli and Monique S.J., Simmonds Phenolic 13. Chinese Academy of science, Flora of Editorial and Terpenoid Constituents from the Sri Lankan Medicinal committees of Flora of China 53.volum [M], Bejing Plant Osbeckia aspera, J of Pharmaceutical Biology, Science press, 53(1) ,138 (1984) 46(3), 154-161 (2008) 14. Su J.D., Oswa T., Kawakishi S., et al., A novel 28. Nichol D. Sl, Daniels H.M., Ira Thabrew M., Grayer R.J., antioxidative synergist isolated from L Simmonds M.S. and Hughes R.D., In vitro studies on the [J], Agri Biol Chem. , 51 , 3449 (1987) immunomodulatory effects of extracts of Osbeckia aspera, J. Ethanopharmacology, 78(1), (2001) 15. Su J.D., Oswa T., Kawakishi S., et al., Antioxidative 39-4 flavanoids isolated from Osbeckia Chinensis L [J], Agri 29. Zeng X., Fang Z. and Ma J., Chemical constituents of Biol Chem., 51, 2801 (1987) Osbeckia chinensis, Article in Chinese J., 16(2), 99-101 16. Su J.D., Oswa T., Kawakishi S, et al.Tannin antioxidants (1991) from Osbeckia Chinensis [J]. Phytochemistry , 27 , 1315 30. Thabrew M.I., Hughes R.D., Gove C.D., Portmann B., (1988) Williams R. and McFarlane I.G., Protective effects of 17. Zeng Xian-meter, Square Zhapu, Ma Jianzhong and Jin against paracetamol-induced liver J.Xenobitica, 25(9), 1995) Jin, Hong Chemical composition of China’s traditional injury, 1009-1017 ( Chinese medicine mischievous Zhi , 16(2) , 99 (1991) 31. M.Ira Thabrew, Christopher D. Gove, Robin D. Hughes, Ian G. McFarlane and Roger Williams, Protective effects 18. Wang Bo and Wang Hao, The temperature far shadow, and Osbeckia Octandra tert so leave the air tank of chemical compostion [J], Days of against galactosamine and -butyl J. Natural product research and development , 12(2) , 45-48 hydroperoxide induced hepatocyte damage. Ethanopharmacology, 49(2), 69-76 (1995) (2001) 32. Thabrew M.I., Joice P.D. and Rajatissa W., A comparative 19. Francisco J.A., Nascimento Agnes M.L. Karlson and Osbeckia Ragnar Elmgren Department of Systems Ecology, study of the efficacy of Pavetta indica and

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octandra in the treatment of liver dysfunction, J.Planta 37. Zeng X., Fang Z. and Ma J., Chemical constituents of medica ., 53(3), 239-41 (1987) Osbeckia chinensis, Article in Chinese J., 16(2) , 99-101 33. Thabrew M.I. and Jayatilaka K.A. P.W., A comparative (1991) study of the beneficial effects of Osbeckia octandra and 38. Jeng-De Su, Toshihiko Osawa, Shunro Kawakishi, Mitsuo Osbeckia aspera in liver dysfunction in rats, The Ceylon Namiki.Tannin antioxidants from Osbeckia chinensis, Journal of Medical Science, 42(1), 1-6 (1999) J.Phytochemistry, 27(5), 1315-1319 (1998) 34. Pathirana Chitra and Bailey Clifford J., Hypoglycemic 39. Syiem D. and Khup P.Z., Study of the Traditionally Used activity of three plant treatments for diabetes mellitus: Medicinal Plant Osbeckia chinensis for Hypoglycemic and artocarpus heterophyllus, Osbeckia Octandra and Anti-hyperglycemic Effects in Mice, J.Pharmaceutical asteracantha longifolia, Galle Medical Journal, 1(1) , 11-17 biology, 44(8) , 613-618 (2006) (1996) 40. Wang Bo, Wang Hao, Wen Yuanying and Hu Changxu, 35. Jayathilaka KAPW, Thabrew M.I., Pathirana C., De Silva The Chemical Constituents of Osbeckia Crinita, J. Natural DGH and Perera DJB, An evaluation of the potency of Product research and development, 12(5), 45-50 (2000) Osbeckia octandra and Melothria maderaspatana as 41. Neshwari Devi M., Biren Singh Kh., Singh S.R., Singh antihepatotoxic agents, Planta Medica , 55(2 ), 137-139 C.B., Deb Lukesh and Dey Amitabh, Antihyperglycemic (1989) effect of Aqueous and Ethanol extract of Aerial part of 36. WANG Hongsheng, WANG Yuehu A., SHI Yana, LI O.nepalensis Hook in Alloxan induced Diabetic rats, Xingyu, LONG Chunlin, Chemical constituents in roots of International Journal of PharmTech Research, 4(1), 233- Osbeckia opipara, China J of Chinese meteria medica, 24 ( 2012) 34(4), 414-418 (2009)

Table-1 Biological activities reported in the Osbeckia genus S. No Plant Name Activity 1 O. aspera 1. Protection by O.aspera against carbon tetrachloride-mediated alterations in microsomal drug metabolizing enzyme activity 26 . 2. Antioxidant activity of O.aspera 27 . 3. Phenolic and Terpenoid Constituents from the Sri Lankan Medicinal Plant O.aspera 28 . 4. In vitro studies on the immunomodulatory effects of extracts of O.aspera 29 . 2 O.crinita 1. The chemical Constituents of O.crinita 30 .

3 O. octandra 1. Protective effects of O.octandra against paracetamol-induced liver injury 31 . 2. Protective effects of O.octandra against galactosamine and tert -butyl hydroperoxide induced hepatocyte damage 32 . 3. A comparative study of the efficacy of Pavetta indica and O.octandra in the treatment of liver dysfunction 33. 4. A comparative study of the beneficial effects of O.octandra and O. aspera in liver dysfunction in rats 34 . 5. Hypoglycemic activity of three plant treatments for diabetes mellitus: artocarpus heterophyllus, O.ctandra and asteracantha longifolia 35 . 6. An evaluation of the potency of O.octandra and Melothria maderaspatana as antihepatotoxic agents 36 . 4 O.opipara 1. Chemical constituents in roots of O.opipara 37 . 5 O.chinensis 1. Chemical constituents of O.chinensis 38 . 2. Tannin antioxidants from O.chinensis 39 . 3. Study of the Traditionally Used Medicinal Plant O.chinensis For Hypoglycemic and Anti- hyperglycemic Effects in Mice 40 . 6 O.nepalensis 1. Antihyperglycemic effect of Aqueous and Ethanol extract of Aerial part of O.nepalensis Hook in Alloxan induced Diabetic rats 41 .

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Table-2 In-vitro antibacterial activity of methanolic extract of O.wynaadensis Bacteria Zone of inhibition (mm) MIC (mg) Gram positive OSW* Ampicillin OSW Ampicillin S. pneumonia 16.0 - 2.5 - B. cereus 14.0 15.0 2.0 5.0 Gram negative OSW Ampicillin OSW Ampicillin A. hydrophila 14.0 - 2.5 - V. cholera 12.0 16.0 2.0 5.0 OSW* : Methanolic extract of O.wynaadensis

Table-3 In- vitro antifungal activity of methanolic extract of O.wynaadensis Zone of inhibition (mm) MIC (mg) Fungi OSW Clotrimazole OSW Candida albicans 11.0 12 2.5 Aspergillus niger 12.0 11.0 2.5 Streptomysis Greusis(mm) 16.0 13.0 1.25 M.purpureus 11.0 15.0 2.5 Aspergillus fumigate 25.0 18.0 1.25 OSW* : Methanolic extract of O.wynaadensis Table 4 In-vitro anti MRSA activity against methanolic extract of O.wynaadensis Zone of inhibition (mm) MIC (mg) Bacteria OSW Ampicillin OSW Ampicillin MRSA 14.0 17.0 2.5 8.0 OSW* : Methanolic extract of O.wynaadensis

Table-5 cytotoxic properties of methanolic extract of O.wynaadensis in human cervical cancer cell line (HeLa) and Murine Embryonic Fibroblasts Cell Line (NH3T3) by MTT assay S. No Cell lines IC in µg/mL 50 1 HeLa 220.3 2 NIH 3T3 92.35

80 100

80 60 60 40 40

20 20 % Growth Inhibition Growth % Inhibition Growth % 0 0 0 1 2 3 0 1 2 3

Log 10 concentration (µg/ml) Log 10 concentration (µg/ml)

Figure-1 Figure-2 MTT assay of O.wynaadensis on HeLa cell line MTT assay of O.wynaadensis on NH3T3 cell line

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